Leishmaniasis is a tropical disease infecting more than 10 million people, causing a spectrum of disease ranging from mild to disfiguring to fatal. The long-term goal of this application is to identify genes responsible for virulence of the parasite as the basic knowledge may permit the development of more sophisticated control strategies. To accomplish this, a variety of genetic methods in Leishmania were developed and applied in the prior grant period. These advances included expression vectors, homologous gene replacement and the creation of gene `knockouts', and functional genetic complementation. This latter method suggested that previously unknown virulence genes could be genetically identified in Leishmania, following the classic paradigm established in prokaryotic pathogenesis studies. Accordingly, susceptible mice were infected with a population of avirulent L. major, previously transfected with a cosmid library made from virulent Leishmania genomic DNA. Unlike the avirulent control, infections were obtained from the transfectant population. Cosmids were recovered from these parasites, and subsequent tests confirmed 10 different cosmids were capable of increasing the infectivity of a completely avirulent line. Current data suggest that these genes affect some step(s) in the mammalian part of the infectious cycle, the stage most relevant to human disease. These VIR cosmids will be characterized in more detail, and additional screens for virulence genes performed, focusing on specific steps of the infectious cycle and using other avirulent lines. Specific aims are: first to determine the step of the life cycle affected by each VIR cosmid using specific assays. Second, the active genes within each cosmid will be mapped and sequenced; its site and mechanisms of action will be sought. Third the genetic basis for VIR gene action will be probed to determine whether they function by complementation or suppression. VIR gene will null mutants will be obtained by gene replacement, incorporating new strategies for obtaining null mutants in essential loci. Developmental regulation will be examined and interactions amongst different genes sought. Fourth, basic studies of the frequency, type and induction of homozygous mutations in Leishmania will be initiated. This information will lead to improved protocols for recovering mutants, which is essential for functional genetic screens.